Blockade of V‐domain immunoglobulin suppressor of T‐cell activation reprograms tumour‐associated macrophages and improves efficacy of PD‐1 inhibitor in gastric cancer

Abstract Background and aims In gastric cancer, the response rate of programmed cell death protein‐1 (PD‐1) inhibitor is far from satisfactory, indicating additional nonredundant pathways might hamper antitumour immunity. V‐domain immunoglobulin suppressor of T‐cell activation (VISTA) has been reported in several malignancies as a novel immune‐checkpoint. Nevertheless, the role of VISTA in gastric cancer still remains obscure. Our purpose is to explore the clinical significance and potential mechanism of VISTA in affecting gastric cancer patients’ survival and immunotherapeutic responsiveness. Methods Our study recruited eight independent cohorts with a total of 1403 gastric cancer patients. Immunohistochemistry, multiplex immunofluorescence, flow cytometry or intracellular flow cytometry, quantitative polymerase chain reaction, western blotting, fluorescence‐activated cell sorting, magnetic‐activated cell sorting, smart‐seq2, in vitro cell co‐culture and ex vivo tumour inhibition assays were applied to investigate the clinical significance and potential mechanism of VISTA in gastric cancer. Results VISTA was predominantly expressed on tumour‐associated macrophages (TAMs), and indicated poor clinical outcomes and inferior immunotherapeutic responsiveness. VISTA+ TAMs showed a mixed phenotype. Co‐culture of TAMs and CD8+ T cells indicated that VISTA+ TAMs attenuated effective function of CD8+ T cells. Blockade of VISTA reprogrammed TAMs to a proinflammatory phenotype, reactivated CD8+ T cells and promoted apoptosis of tumour cells. Moreover, blockade of VISTA could also enhance the efficacy of PD‐1 inhibitor, suggesting that blockade of VISTA might synergise with PD‐1 inhibitor in gastric cancer. Conclusions Our data revealed that VISTA was an immune‐checkpoint associated with immunotherapeutic resistance. Blockade of VISTA reprogrammed TAMs, promoted T‐cell‐mediated antitumour immunity, and enhanced efficacy of PD‐1 inhibitor, which might have implications in the treatment of gastric cancer.


INTRODUCTION
Gastric cancer is a major global health burden. 1 Worldwide, gastric cancer is the fifth most diagnosed cancer, with over 1,000,000 estimated new cases annually.Due to its covert early symptoms, gastric cancer is frequently diagnosed at advanced-stage, making it the fourth most common cause of malignancy-associated deaths. 2 In recent years, although progress has been made in adjuvant treatment, the prognosis of advanced stage gastric cancer patients still remains dismal and requires to be improved. 3tepping into the 21st century, cancer immunotherapy with immune-checkpoint blockade (ICB) has emerged as a powerful weapon against cancer and led to important clinical advances. 4Antibodies targeting immune-checkpoint receptors of B7 family, cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) and programmed cell death protein-1 (PD-1), have shown durable response in a series of previously refractory malignancies, which are considered as a breakthrough in the treatment of cancer. 4,5In 2021, nivolumab (Opdivo, Bristol-Myers Squibb Company) was approved as first-line treatment agents for advanced or metastatic gastric cancer and gastroesophageal junction cancer in the United States and the People's Republic of China.Despite this success, the response rate of ICB was generally less than 30%, and single-agent PD-1 inhibitors reported response rates of only 10%-17% for unselected patients with metastatic gastroesophageal cancer, 6,7 indicating additional non-redundant immunomodulation pathways might attenuate antitumour immunity in gastric cancer.
As an immune-checkpoint of B7 family. 8,9V-domain immunoglobulin suppressor of T-cell activation (VISTA) is expressed on several types of immune cells, including macrophages, dendritic cells (DCs), naïve CD4 + T cells and Foxp3 + CD4 + regulatory T (T reg ) cells, with the highest expression found on myeloid cells. 8,10Different from PD-1 or CTLA-4, which controls T-cell-mediated immunity and antagonizes T-cell receptor signalling, 11,12 VISTA plays a more profound role in modulating myeloid cell-mediated immune responses. 13Therefore, VISTA could serve as a potential immunotherapeutic target. 14,15Phase I/II clinical trials of VISTA inhibitors (JNJ-61610588; CI-8993; CA-170) are currently ongoing. 16Nevertheless, knowledge about the role of VISTA in gastric cancer is still limited.Whether VISTA reprograms macrophages and indirectly controls tumour-specific T-cell responses in gastric cancer has not been elucidated, either.
Our study revealed a potential role of VISTA in modulating the phenotype of tumour-associated macrophages (TAMs) and orchestrating immunotherapeutic resistance.Blockade of VISTA abolished the immunosuppressive function of TAMs and led to a T-cell-effective tumour microenvironment that drove an efficient antitumour response.We propose VISTA as a new immunotherapeutic target in gastric cancer.

Immunohistochemistry and multiplex immunofluorescence
The construction of tissue microarray (TMA) was documented (Supporting Information S1).The TMAs were heated at 60 • C for 6 h, then immersed in xylene (three times, 15 min each) and alcohol (100%, 95%, 85%, 75%), and rinsed with Tris Buffered Saline with Tween 20 (TBST) (three times, 10 min each).Antigen retrieval was performed with Ethylene Diamine Tetraacetic Acid (EDTA) or Citrate antigen retrieval solution (ZSGB-BIO).Endogenous peroxidase was blocked by endogenous peroxidase blocking buffer (ZSGB-BIO) at 37 • C for 30 min.Then, Normal Goat Serum (ZSGB-BIO) was used to eliminate nonspecific reactions.Subsequently, the TMAs were incubated with primary antibody (Table S2) at 4 • C overnight.Negative controls were treated identically with the primary antibody abandoned.After washing with TBST (three times, 10 min each), the TMAs were incubated with Horseradish Peroxidase (HRP)-conjugated secondary antibody (ZSGB-BIO) at 37 • C for 20 min.For immunohistochemistry (IHC) staining, the TMAs were subsequently stained with 3,3'-diaminobenzidine (DAB), counterstained with haematoxylin, then dehydrated and finally mounted with neutral resins and coverslip.For multiplex immunofluorescence (MxIF) staining, after incubation with HRP-conjugated secondary antibody, the TMAs were immersed in Neon TSA fluorescent dye at room temperature for 10 min and washed with TBST (three times, 3 min each).The TMA slides underwent repeated steps mentioned above since antigen retrieval to staining with fluorescent dye.Then, the TMA slides were stained with 4',6-diamidino-2-phenylindole (DAPI) at room temperature for 5 min.Eventually, the TMAs were sealed with anti-fluorescence quenching sealant and coverslip.

Analysis of VISTA expression in gastric cancer
Expression of VISTA protein was scored in a blinded fashion.A detailed description of the IHC scoring system, together with representative images, scoring results and prognostic power analysis, was provided (Supporting Information S2 and Figures S2-S4).

2.4
Fluorescence-activated cell sorting, magnetic-activated cell sorting and

Quantitative polymerase chain reaction
TAMs and tumour-infiltrating CD8 + T cells were isolated from fresh gastric cancer tissues by human CD14 and CD8 nanobeads (MojoSort, BioLegend), respectively.Total RNA from TAMs, tumour-infiltrating CD8 + T cells and AGS cell line was extracted by FastPure Total RNA Isolation Kit (Vazyme Biotech).RNA quantity and quality were determined with use of DS-11 Spectrophotometer (DeNovix).RNA was transcribed into cDNA by PrimeScript RT reagent Kit (TaKaRa).Quantitative polymerase chain reaction (qPCR) was performed on QuantStudio5 (Applied Biosystems) with use of SYBR Green dye (TaKaRa).The primers used in this study were also listed (Table S4).At ).TCGA-STAD was a public gastric cancer database including 407 gastric cancer patients, 375 of whom had comprehensive mRNA expression data (Figure S1), downloaded from National Cancer Institute GDC Data Portal (https://portal.gdc.cancer.gov/).(B) In Expansion Dataset #1 (Samsung Medical Center Sungkyunkwan University [SKKU-SMC]), the patients stratified in VSIR high subgroup had significantly higher rate of progressive disease (PD)/stable disease (SD), compared with those in VSIR low subgroup after pembrolizumab-based immunotherapy.In SKKU-SMC cohort, gastric cancer patients were administered with a 30-min intravenous infusion of pembrolizumab 200 mg every 3 weeks until documented disease progression, unacceptable toxicity, or up to 24 months.Tumour tissues were obtained before initiation of immunotherapy. 19SKKU-SMC was a public gastric cancer immunotherapy database including 61 gastric cancer patients, 45 of whom had comprehensive mRNA expression data (Figure S1), downloaded from TIDE (http://tide.dfci.harvard.edu/).(C) In Expansion Dataset #1 (SKKU-SMC), the patients stratified in VSIR high subgroup had poorer overall survival (OS) than those in VSIR low subgroup.SKKU-SMC was a public gastric cancer immunotherapy database including 61 gastric cancer patients, 43 of whom had both mRNA data and survival data (Figure S1).Significance values were determined by Pearson's χ 2 test (B) or log-rank test (C).The cut-off value for classification of VSIR high subgroup and VSIR low subgroup was the median value.CR, complete response; CTLA-4, cytotoxic T-lymphocyte-associated protein-4; HAVCR2, hepatitis A virus cellular receptor 2; KLRC1, killer cell lectin like receptor C1; LAG-3, lymphocyte activating gene-3; NKG2A, natural killer group 2 member A; PD-L1, programmed cell death 1-ligand 1; PD-1, programmed cell death protein-1; PR, partial response; TIGIT, T-cell immunoreceptor with Ig and ITIM domains; TIM-3, T-cell immunoglobulin domain and mucin domain-3; VISTA, V-domain immunoglobulin suppressor of T-cell activation; VSIR, V-set immunoregulatory receptor.least three independent experiments were repeated for one sample.Relative transcript levels were estimated by means of ΔΔCt method.

Western blot
RIPA lysis buffer (Epizyme) was used to lyse cells.After centrifugation, the supernatant was collected in a clean tube.BCA Protein Assay Kit (Epizyme) was applied to quantify protein concentrations.Subsequently, loading buffer (Epizyme) was mixed with supernatant and heated.Samples of equal amounts were separated by 10% PAGE Gel Fast Preparation Kit (Epizyme) and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore).PVDF membranes were blocked with skimmed milk powder (Servicebio) dissolved in TBST at room temperature for 2 h.The membranes were incubated with primary antibodies against VISTA (1:1000, CST) and GAPDH (1:10 000, CST; Table S2) at 4 • C overnight.GAPDH was used as control.After incubation with secondary antibodies (1:5000, Servicebio), the signals were visualised by the ECL system (Clinx Science Instruments).Experiments were performed in triplicate.

Ex vivo tumour inhibition assay
Single-cell suspensions derived from 10 freshly resected gastric cancer tissues were randomly divided into four treatment groups (Figure 7A with α-VISTA antibody (15 μg/mL, R&D Systems) or IgG 2B (15 μg/mL, R&D Systems) for 12 h at 37 • C.After overnight culture, the cells were harvested for phenotype analysis by FC/ICFC.Detailed information about the antibodies was supplemented (Table S2).

Flow cytometry and intracellular flow cytometry
Freshly resected gastric cancer tissues were collected, digested and incubated with GolgiStop (BD Biosciences) at 37 • C for 2 h.Then, the single-cell suspensions were lysed with lysing buffer (BD Biosciences), protected from light, at room temperature for 15 min.The cells were stained with FVS510 (BD Biosciences) to determine live/dead cells.Afterwards, human Fc Block (BD Biosciences) was applied to block Fc receptor.Cells were stained for surface markers at 4 • C for 30 min and protected from light.If necessary, cells were fixed and permeabilized with Fixation and Permeabilization Solution (BD Biosciences) at 4 • C for 20 min.Intracellular cytokine staining was performed at 4 • C for 30 min and protected from light.Stained cells were ultimately washed and resuspended in stain buffer (BD Biosciences).Flow cytometry (FC) or intracellular flow cytometry (ICFC) was performed by FACSCelesta or FAC-SAria III (BD Biosciences).Data were analysed by FlowJo (Tree Star).S3).p < .05 was defined as statistically significant.

VSIR shows distinct expression pattern and predicts inferior immunotherapeutic responsiveness in gastric cancer
In Discovery Dataset (TCGA-STAD), we investigated the expression pattern of eight immune-checkpointassociated genes in gastric cancer: VSIR (VISTA), 26 HAVCR2 (TIM-3), 27 TIGIT (TIGIT), 28 KLRC1 (NKG2A), 29 CD274 (PD-L1), 30 LAG3 (LAG-3), 31 CTLA4 (CTLA-4) 4 and PDCD1 (PD-1), 32 which were previously reported to be promising targets for immunotherapy.We found that the expression pattern of VISTA-encoding gene VSIR was distinguished from those of other genes (Figure 1A), indicating VSIR (VISTA) might mediate an additional nonredundant immune-checkpoint pathway in gastric cancer.Consequently, we included Expansion Dataset #1 from SKKU-SMC and inspected the association between VSIR expression and patient responsiveness to pembrolizumab-based immunotherapy (Figure S1).In SKKU-SMC cohort, gastric cancer patients were administered with pembrolizumab.Tumour tissues were obtained before initiation of immunotherapy. 19Of note, we found after the application of pembrolizumab, the patients stratified in VSIR high subgroup had significantly higher rate of progressive disease (PD) or stable disease (SD), compared with those in VSIR low subgroup (Figure 1B).Furthermore, the patients stratified in VSIR high subgroup also underwent significantly poorer overall survival (OS) than those in VSIR low subgroup after pembrolizumab-based immunotherapy (Figure 1C).Conclusively, these results indicated that high expression of VSIR could predict inferior immunotherapeutic responsiveness in gastric cancer.

Expression of VISTA correlates with adverse survival outcomes in gastric cancer
IHC showed that the expression of VISTA in tumour tissues was significantly higher than that in peritumour tissues (Figure S2).Moreover, VISTA was predominantly expressed in tumour stroma, whereas malignant epithelial showed almost no expression of VISTA (Figure S3).Consequently, we focused on the expression of VISTA in tumour tissues in the following study (Figure S4).Interestingly, we found that higher proportion of stage II and stage III patients were stratified into VISTA high subgroup, compared with stage I patients (Figure 2A), indicating that VISTA was possibly associated with tumour progression.The patients stratified in VISTA high subgroup had significantly poorer OS and disease-free survival (DFS) than the patients in VISTA low subgroup (Figure 2B,C).Notably, high expression of VISTA predicted poorer OS and DFS in any tumour-node-metastasis (TNM) stage (Figure 2D-I).
These results showed that high VISTA expression indicated adverse survival outcomes in gastric cancer, which was independent of TNM stage.Furthermore, according to univariate analysis, size, Lauren classification, TNM stage and VISTA expression showed significant prognostic value in patient OS, while sex, size, Lauren classification, TNM stage and VISTA expression for DFS (Table 1).However, multivariate analysis showed that age, TNM stage and VISTA expression were significant variables for OS, and sex, Lauren classification, TNM stage and VISTA expression for DFS (Table 1).To compare the prognostic power between these significant variables, we applied time-dependent area under the curve (AUC).Interestingly, TNM stage showed highest AUC in predicting either OS or DFS, and VISTA expression was inferior to TNM stage but superior to age, sex or Lauren classification (Figure S5).Conclusively, these results emphasised the clinical significance of VISTA and characterised VISTA as an independent adverse prognosticator in gastric cancer.

VISTA is predominantly expressed on TAMs in gastric cancer
Since VISTA might inhibit antitumour immunity, we subsequently sought for the source of VISTA in gastric cancer.According to the scRNA-seq data from Expansion Dataset #3 (GSE134520), 20 we found VSIR was expressed on several kinds of cells, especially myeloid cells including macrophages, monocytes and DCs (Figure S6).Another scRNA-seq database PKU-scDVA 21 also indicated that VSIR was predominantly expressed on monocytes/macrophages in gastric cancer (Figure 3A,B).Consequently, we collected 12 fresh gastric cancer samples from FDU-ZSH (EXPC Arm A), and performed FC to validate the distribution of VISTA (Figure 3C).We found that VISTA was primarily expressed on TAMs (Figures 3D and  S7).MxIF also verified the co-expression of VISTA and CD68 in gastric cancer (Figure 3E).Since previous studies reported VISTA could also be expressed by CD8 + T cells and cancer cells, 33 or expressed VISTA may be translocated onto the cell surface, where it contributes to suppression of T-cell activity. 34VISTA can also be shed off the cell surface and secreted (40 kDa soluble VISTA). 35,36Furthermore, we studied the VSIR mRNA level in TAMs to immunofluorescence (MxIF) was performed to validate the co-expression of VISTA and CD68 in gastric cancer.White arrowheads show VISTA + TAMs.Haematoxylin and eosin (H&E) staining was also applied with the same tissue microarray (TMA).Scale bar represents 50 μm.FDU-ZSH (T19-0097) was our own dataset, including 60 gastric cancer patients (Figure S1).confirm that VISTA gene is expressed in these cells and that this protein did not come as a secreted protein from gastric cancer cells.Western blotting (WB) was also performed in TAMs to see what kind of VISTA expressed.According to qPCR, we found that TAMs showed significantly higher mRNA level of VSIR compared with CD8 + T cells or gastric cancer cells.According to WB, we also found that TAMs showed significantly higher expression of VISTA compared with CD8 + T cells or gastric cancer cells.Additionally, we found that TAMs expressed 55 kDa VISTA (Figure S8).Consequently, these results indicated that VISTA was expressed by TAMs and might serve as a receptor other than an extracellular domain proteolytically shed off the surface of other cells.Association between the expression of VISTA and CD68 was also validated in gastric cancer patient cohort (Figure 3F).Collectively, these findings suggested that VISTA was predominantly expressed on TAMs in gastric cancer.

VISTA + TAMs show a mixed phenotype
To inspect the characteristics of VISTA + TAMs, we collected eight fresh gastric cancer samples from FDU-ZSH (EXPC Arm B), and performed FACS to sort eight pairs of VISTA + TAMs and VISTA − TAMs.After quality control (QC), three pairs of VISTA + TAMs and VISTA − TAMs were subsequently performed with smart-seq2 (Figure 4A).VISTA + TAMs expressed significantly higher expression of genes such as C1QB, STAB1, C1QA, FCN1 and RAB31 (Figure 4B), and showed different gene profile compared with VISTA − TAMs (Figure 4C).Notably, C1QA + macrophage simultaneously resembled the signatures for TAMs and showed both M1 and M2-like phenotype, 37 while FCN1 + monocyte-like precursors might develop into C1QC + TAMs, which exhibited enriched complement activation, antigen processing and presentation pathways.And these cells could interact with other immune cells, especially T-cell subsets. 21,38However, STAB1 (Stabilin-1) was an M2-like macrophage marker indicating immunosuppressive phenotype. 39,40The GO analysis indicated that the up-regulated genes in VISTA + TAMs might be associated with immune response, innate immune response and inflammatory response (Figure 4D).Moreover, we found that the gene expression profile of transmembrane receptors, cytokines and transcription factors characterised VISTA + TAMs as a specific subset of TAM populations that exhibited a mixed phenotype of M1 and M2-like TAMs (Figure 4E,F), which was consistent with a previous study indicating M1 and M2 signatures did not necessarily exclude each other and often coexisted in TAMs. 41FC/ICFC analysis with fresh gastric cancer samples (FDU-ZSH EXPC Arm C) also validated that VISTA + TAMs exhibited significantly higher expression of M2-like marker CD163 and CD206, as well as M1-like marker human leukocyte antigen (HLA)-DR and CD11c (Figure 4G).Moreover, we found that VISTA + TAMs also showed higher expression of tumour necrosis factor-α (TNF-α), interleukin (IL)-12, programmed cell death 1ligand 1 (PD-L1), arginase (Arg)-1 and latency-associated peptide (LAP)/transforming growth factor (TGF)-β, compared with VISTA − TAMs (Figure 4H).Conclusively, these findings indicated that the phenotype of VISTA + TAMs might be more complicated than the classical dichotomous phenotypes of M1 or M2-like TAMs.

Blockade of VISTA reprograms TAMs in gastric cancer
Subsequently, we isolated TAMs from gastric cancer tissues after VISTA blockade or isotype treatment, and applied smart-seq2 to inspect the impact of VISTA blockade on TAMs (Figure 5A).Interestingly, 651 DEGs were detected (Table S5).According to these DEGs, we constructed a VISTA + TAM signature.Notably, higher VISTA + TAM signature indicated significantly poorer OS of VISTA + TAMs versus VISTA − TAMs were detected in FDU-ZSH EXPC (Arm B). (D) The Gene Ontology (GO) analysis based on the DEGs of VISTA + TAMs versus VISTA − TAMs indicated that the up-regulated genes in VISTA + TAMs might be associated with immune response, innate immune response and inflammatory response, with use of FDU-ZSH EXPC (Arm B). (E and F) The gene profile of transmembrane receptors, cytokines and transcription factors, based on the DEGs of VISTA + TAMs versus VISTA − TAMs, characterised VISTA + TAMs as a specific subset of TAM populations that exhibited a mixed phenotype of M1 and M2-like TAMs.(G) In FDU-ZSH EXPC (Arm C), FC analysis with 10 fresh GC samples validated that VISTA + TAMs exhibited significantly higher expression of M2-like marker CD163 and CD206, as well as M1-like marker human leukocyte antigen (HLA)-DR and CD11c.FDU-ZSH EXPC (Arm C) was our own dataset and contained 10 GC patients (Figure S1).(H) In FDU-ZSH EXPC (Arm C), VISTA + TAMs showed higher expression of tumour necrosis factor-α (TNF-α), interleukin (IL)-12, programmed cell death 1-ligand 1 (PD-L1), arginase (Arg)-1 and latency-associated peptide (LAP)/transforming growth factor (TGF)-β (marginally insignificant), compared with VISTA − TAMs. in TCGA-STAD database as well as ACRG (GSE62254) database (Figure S9).3][44][45][46][47][48][49][50] Blockade of VISTA activated the genes associated with immune responses (Figure 5D), especially the genes modulating proinflammatory phenotype of macrophages and CD8 + T-cell activation (Figure 5E).By means of FC/ICFC, we also validated that blockade of VISTA significantly up-regulated the expression of TNFα and IL-12, yet down-regulated the expression of Arg-1 and LAP/TGF-β within TAMs (Figures 5F and S10).Of note, blockade of VISTA could not interfere with PD-L1 expression on TAMs (Figure S10), indicating VISTA blockade might be non-overlapping with PD-1/PD-L1 pathways, which was consistent with a previous study. 51Conclusively, these findings suggested that blockade of VISTA might reprogram TAMs to a proinflammatory phenotype.

VISTA + TAMs attenuate effective function of CD8 + T cells
Since VISTA + TAMs might interfere with CD8 + T-cell function (Figure 5E), we subsequently sought to inspect the potential impact of VISTA + TAMs on CD8 + T cells.Based on FDU-ZSH (T13-564) cohort (Figure 6A), we found that infiltration of CD68 + TAMs was positively correlated with CD8 + T cells, regardless of VISTA expression (Figure 6C).Notably, increased infiltration of CD8 + T cells could predict better OS and DFS, except for the VISTA high CD68 high patients (Figure 6D), indicating VISTA + TAMs might attenuate CD8 + T-cell function.According to the results from FC/ICFC and MxIF (Figure 6B), we found that higher infiltration of VISTA + TAMs was associated with decreased expression of interferon-γ (IFN-γ) and granzyme B (GzmB) within CD8 + T cells (Figure 6E,F).In TAM/T cell co-culture sys-tem, blockade of VISTA mitigated the immunosuppressive effect of TAMs on CD8 + T cells (Figure 6G,H).Collectively, these results indicated that VISTA + TAMs could possibly attenuate effective function of CD8 + T cells.

Blockade of VISTA reactivates effective function of CD8 + T cells and enhances efficacy of PD-1 inhibitor
Finally, we established an ex vivo tumour inhibition assay to investigate the potential impact of VISTA and/or PD-1 blockade in gastric cancer (Figure 7A).Interestingly, we found blockade of PD-1 showed increased apoptosis of tumour cells in the tumours infiltrated with VISTA low TAMs, rather than VISTA high TAMs.Similarly, after the application of camrelizumab, elevated expression of IFNγ and GzmB within CD8 + T cells was observed in the tumours with VISTA low TAMs rather than VISTA high TAMs (Figure 7C,D).These results indicated that VISTA + TAMs were associated with reduced efficacy of PD-1 inhibitor camrelizumab on promotion of tumour cell apoptosis and reactivation of CD8 + T-cell-effective function.Notably, blockade of VISTA showed increased apoptosis of tumour cells (Figure 7E), as well as increased expression of IFN-γ, GzmB and Perforin within CD8 + T cells (Figure 7F-H).Moreover, combination of VISTA blockade and PD-1 inhibitor camrelizumab showed more significant apoptosis of tumour cells and reactivation of CD8 + T cells, compared with VISTA blockade or camrelizumab alone, suggesting a potential synergistic effect of VISTA blockade and PD-1 inhibitor camrelizumab in gastric cancer.To figure out the relation between promotion of tumour cell apoptosis and reactivation of CD8 + T cells, we established another ex vivo tumour inhibition assay with CD8 + T cells depleted (Figure 7B).The antitumour effect of VISTA blockade was abolished when CD8 + T cells were depleted, while the single-cell suspensions with CD8 + T cells depletion alone did not show significant decrease in tumour cell apoptosis compared with those without CD8 + bovine serum (FBS; Gibco) for 12 h at 37 • C. Percoll (Cytiva) was subsequently applied to separate mononuclear cells.IgG 2B -treated TAMs and α-VISTA antibody-treated TAMs were isolated by CD14 magnetic-activated cell sorting (MACS) kit, and performed with smart-seq2.FDU-ZSH EXPC (Arm D) was our own dataset and contained 5 GC patients (Figure S1).(B and C) Differentially expressed genes (DEGs) of α-VISTA antibody-treated TAMs versus IgG 2B -treated TAMs were detected within FDU-ZSH EXPC (Arm D). (D and E) In FDU-ZSH EXPC (Arm D), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that blockade of VISTA activated the genes associated with immune responses, especially the genes modulating proinflammatory phenotype of macrophages and CD8 + T-cell activation.(F) According to flow cytometry (FC)/intracellular flow cytometry (ICFC) (FDU-ZSH EXPC Arm G), blockade of VISTA significantly up-regulated the expression of interleukin (IL)-12 and tumour necrosis factor-α (TNF-α), yet down-regulated the expression of arginase (Arg)-1 and latency-associated peptide (LAP)/transforming growth factor (TGF)-β within TAMs.FDU-ZSH EXPC (Arm G) was our own dataset, including 10 GC patients (Figure S1).Significance values were determined by Wilcoxon matched-pairs signed rank test (F).*p < .05 and **p < .01.
T cells depletion (Figure 7I), indicating blockade of VISTA might eventually reactivate effective function of CD8 + T cells and thus promote tumour cell apoptosis.Conclusively, these data suggested that blockade of VISTA could possibly reactivate effective function of CD8 + T cells and promote tumour cell apoptosis, and enhance efficacy of PD-1 inhibitor in gastric cancer.

DISCUSSION
In recent years, several studies have reported VISTA as a potential prognosticator.Nevertheless, the conclusions remain controversial.In triple-negative breast cancer (TNBC), VISTA indicated favourable prognosis and high infiltration of immune cells. 52,53However, such published studies on TNBC were primarily bioinformatic ones and lacked further experiments to elucidate the distribution of VISTA or the mechanism of VISTA-associated immuno-oncology.In early-stage esophageal adenocarcinoma, Loeser et al. found that VISTA positive tumours showed an obvious survival advantage compared with VISTA-negative ones. 54However, the authors were concentrated on the expression of VISTA within tumourinfiltrating T-lymphocytes, and did not discuss VISTA on other cell types.Contrarily, Kuklinski et al. suggested that expression of VISTA was a negative prognosticator in primary cutaneous melanoma. 55To date, few studies have reported VISTA in gastric cancer.The clinical significance and underlying mechanism of VISTA-associated immuno-oncology in gastric cancer still remain elusive.
VISTA is reported to play a profound role in the modulation of immune responses. 8,56Myeloid cells show the predominant expression of VISTA, but lymphoid cells such as T lymphocytes also express VISTA. 8,56VISTA could serve as both receptor and ligand. 8,56Receptors of VISTA require further investigation.One of the VISTA ligands is V-set and immunoglobulin domain containing-3 (VSIG-3). 57Another ligand for VISTA is galectin-9. 36Galectin-9 can interact with VISTA on the surface of T cells, which results in T-cell-programmed death. 36In addition, recent study has reported that anti-VISTA could boost antigen presentation pathways yet reduce myeloid cell-mediated immunosuppression, indicating VISTA might play a crucial part in regulating immunosuppression function of myeloid cells. 58n current study, we observed that VSIR showed a unique expression pattern distinct from other immunecheckpoint genes in gastric cancer, which indicated that not all gastric cancer might have the same immunothera-F I G U R E 6 V-domain immunoglobulin suppressor of T-cell activation (VISTA + ) tumour-associated macrophages (TAMs) attenuate effective function of CD8 + T cells.(A) In Zhongshan Hospital Fudan University (FDU-ZSH) (T13-564) cohort, we performed immunohistochemistry (IHC) staining for VISTA, CD68 and CD8.FDU-ZSH (T13-564) was our own gastric cancer (GC) database including 496 GC patients, 473 of whom had resectable disease and comprehensive survival data (Figure S1).Eighteen patients had dot loss during the IHC staining, and finally 455 patients were assessable for VISTA, CD68 and CD8 in FDU-ZSH (T13-564) cohort.The cut-off values for VISTA (6/HPF), CD68 (65/HPF) and CD8 (73/HPF) were median values according to IHC scores, respectively.(B) In FDU-ZSH (T19-0097) cohort, we performed flow cytometry (FC)/intracellular flow cytometry (ICFC) to analyse the phenotype of CD8 + T cells in tumour tissues, and we also conducted immunofluorescence (IF) staining for VISTA and CD68 with the tissue microarray (TMA) derived from the corresponding formalin-fixed and paraffin-embedded (FFPE) samples.FDU-ZSH (T19-0097) was our own dataset including 60 GC patients (Figure S1).(C) In FDU-ZSH (T13-564) cohort, infiltration of CD8 + T cells and CD68 + TAMs were analysed in VISTA high and VISTA low patients.The infiltration of CD8 + T cells were positively correlated with CD68 + TAMs, regardless of VISTA expression.(D) In VISTA high CD68 high patients, higher infiltration of CD8 + T cells could not predict better overall survival (OS) or disease-free survival (DFS).However, higher infiltration of CD8 + T cells could significantly predict better OS and DFS in other patients.The cut-off values for CD68 and CD8 were the median value, respectively.(E) Multiplex immunofluorescence (MxIF) staining for VISTA and CD68 with the FDU-ZSH (T19-0097) TMA.Red colour represents VISTA, green colour represents CD68 and blue colour represents DAPI.Haematoxylin and eosin (H&E) staining for the T19-0097 TMA was also shown.Scale bar represents 50 μm.(F) According to the results from FC/ICFC and IF staining, which were performed within FDU-ZSH (T19-0097), principal component analysis (PCA) indicated that higher infiltration of VISTA + TAMs was associated with decreased expression of interferon-γ (IFN-γ) and granzyme B (GzmB) within CD8 + T cells, while expression of Perforin showed marginal insignificance.Each symbol represents an individual patient.(G) Co-culture with IgG 2B -treated TAMs could lead to decreased expression of IFN-γ and GzmB within tumour-infiltrating CD8 + T cells, while co-culture with α-VISTA-treated TAMs showed restored cytotoxicity of CD8 + T cells.Data are expressed as mean ± standard deviation.FDU-ZSH Experimental Cohort (EXPC) (Arm E) was our own dataset and contained 5 GC patients (Figure S1).(H) Co-culture with IgG 2B -treated TAMs could lead to decreased expression of IFN-γ and GzmB within peripheral CD8 + T cells, while co-culture with α-VISTA-treated TAMs showed restored cytotoxicity of CD8 + T cells.Data are expressed as mean ± standard deviation.FDU-ZSH EXPC (Arm F) was our own dataset and contained 5 GC patients (Figure S1).Significance values were determined by Spearman's correlation test (C), log-rank test (D), Student's t-test (F [Perforin, PD-1 and TIM-3]), Mann-Whitney U-test (F [IFN-γ, GzmB, CD107a, CTLA-4 and LAG-3]) and RM one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test (G and H).CTLA-4, cytotoxic T-lymphocyte-associated protein-4; LAG-3, lymphocyte activation gene-3; MACS, magnetic-activated cell sorting; PD-1, programmed cell death protein-1; TIM-3, T-cell immunoglobulin domain and mucin domain-3.

F I G U R E 7
Blockade of V-domain immunoglobulin suppressor of T-cell activation (VISTA) enhances effective function of CD8 + T cells and promotes efficacy of programmed cell death protein-1 (PD-1) inhibitor.(A) An ex vivo tumour inhibition assay was established to peutic responsiveness.Furthermore, we found that VISTA was preferentially expressed on TAMs in gastric cancer.Since previous studies reported VISTA could also be expressed by CD8 + T cells and cancer cells, 33 or expressed VISTA may be translocated onto the cell surface, where it contributes to suppression of T-cell activity. 34VISTA can also be shed off the cell surface and secreted (40 kDa soluble VISTA). 35,36Furthermore, we studied the VSIR mRNA level in TAMs to confirm that VISTA gene was expressed in these cells and that this protein did not come as a secreted protein from gastric cancer cells.WB was also performed in TAMs to see what kind of VISTA expressed.According to qPCR, we found that TAMs showed significantly higher mRNA level of VSIR compared with CD8 + T cells or gastric cancer cells.According to WB, we also found that TAMs showed significantly higher expression of VISTA compared with CD8 + T cells or gastric cancer cells.Additionally, we found that TAMs expressed 55 kDa VISTA.Consequently, these results indicated that VISTA was expressed by TAMs and might serve as a receptor other than an extracellular domain proteolytically shed off the surface of other cells.According to FACS, smart-seq2 and FC/ICFC, VISTA + TAMs showed a mixed phenotype, more complicated than the classical dichotomous phenotypes of M1 or M2-like TAMs, which might explain the paradoxical effect of VISTA in various malignancies to some extent.In view of recent studies on macrophage functions, growing evidence has proven that M1 and M2 phenotypes do not necessarily exclude each other but usually coexist.The mixed phenotype of macrophages relies on the balance of activatory and inhibitory activities, as well as the corresponding microenvironment. 41ur data suggested that these VISTA + TAMs could attenuate effective function of CD8 + T cells and orchestrate immunotherapeutic resistance to PD-1 inhibitor.Blockade of VISTA could reprogram TAMs to a proinflammatory phenotype, reactivate effective function of CD8 + T cells, promote tumour cell apoptosis, and enhance efficacy of PD-1 inhibitor.These findings highlighted the significance of dual blockade of immune-checkpoints, such as PD-1/PD-L1 and/or VISTA, might provide substantial clinical benefit for patients with gastric cancer.
Although our current study was a retrospective and exploratory one, which was primarily based on clinical specimens, we advocated for future studies to elucidate the mechanism of VISTA-associated immunotherapeutic resistance, and validate whether VISTA could be targeted to reactivate antitumour responses in gastric cancer, within the framework of international, multi-centred randomised clinical trials.
T cells.Blockade of VISTA reprogrammed TAMs to a proinflammatory phenotype, reactivated effective function of CD8 + T cells, promoted tumour cell apoptosis, and enhanced efficacy of PD-1 inhibitor.This study highlighted the clinical significance of VISTA and also offered possibilities to improve the immunotherapeutic strategy of ICB in gastric cancer.

A U T H O R C O N T R I B U T I O N S
Yifan Cao was responsible for sample collection, experiments and data acquisition, analysis and interpretation, and drafting of the manuscript.Kuan Yu and Zihao Zhang were responsible for technical and material support, sample collection and western blot.Yun Gu was responsible for material support and contact with Dr. Jeeyun Lee (Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine).Yichao Gu was responsible for material support.Weijuan Zhang and Wandi Li were responsible for flow cytometry analysis.Zhenbin Shen and Jiejie Xu were responsible for study concept and supervision.Jing Qin was responsible for study concept, design, supervision and obtaining funding.All the authors read and approved the final manuscript.

F I G U R E 2
High expression of V-domain immunoglobulin suppressor of T-cell activation (VISTA) predicts poor survival outcomes in gastric cancer.(A) In Internal Validation Dataset (Zhongshan Hospital Fudan University [FDU-ZSH] T13-564), higher proportion of stage II and stage III patients were stratified into VISTA high subgroup than stage I patients.FDU-ZSH (T13-564) was our own gastric cancer database including 496 gastric cancer patients, 473 of whom had resectable disease and comprehensive survival data (Figure S1).(B-I) In Internal Validation Dataset (FDU-ZSH T13-564), high expression of VISTA predicted poor overall survival (OS) and disease-free survival (DFS) in any tumour-node-metastasis (TNM) stage.Significance values were determined by Pearson's χ 2 test followed by Bonferroni multiple comparison test (A) or log-rank test (B-I).*p < .05,***p < .001,and n.s.refers to not significant.The cut-off value for classification of VISTA high subgroup and VISTA low subgroup was the median value.

F
I G U R E 3 V-domain immunoglobulin suppressor of T-cell activation (VISTA) is predominantly expressed on tumour-associated macrophages (TAMs) in gastric cancer.(A and B) According to Expansion Dataset #4 (Peking University-short for single-cell RNA-seq data visualisation and analysation [PKU-scDVA]), VSIR was potentially expressed on monocytes/macrophages in gastric cancer.PKU-scDVA was a public single-cell RNA-seq database and contained 10 gastric cancer patients' single-cell RNA-seq data (Figure S1), which were available at scDVA online website (http://panmyeloid.cancer-pku.cn/).(C and D) Twelve fresh gastric cancer samples (Zhongshan Hospital Fudan University [FDU-ZSH] Experimental Cohort [EXPC] Arm A) were collected and performed with flow cytometry (FC)/intracellular flow cytometry (ICFC).VISTA was primarily expressed on TAMs.Data in panel (D) are expressed as mean ± standard deviation.FDU-ZSH EXPC (Arm A) was our own dataset including 12 gastric cancer patients.Fresh gastric cancer samples were collected from these 12 patients and performed with FC to investigate VISTA expression within different cell types (Figure S1).(E) In FDU-ZSH (T19-0097) cohort, multiplex (F) In FDU-ZSH (T13-564) cohort, high expression of VISTA was correlated with increased infiltration of CD68 + TAMs.Data in panel (F) are expressed as mean ± standard deviation.Scale bar represents 50 μm.Significance values were determined by Kruskal-Wallis test followed by Dunn's multiple comparisons test (D) and Student's t-test (F).**p < .01,***p < .001.UMAP, uniform manifold approximation and projection; VSIR, V-set immunoregulatory receptor.F I G U R E 4 V-domain immunoglobulin suppressor of T-cell activation (VISTA + ) tumour-associated macrophages (TAMs) show a mixed phenotype in gastric cancer.(A) Eight fresh gastric cancer samples (Zhongshan Hospital Fudan University [FDU-ZSH] Experimental Cohort [EXPC] Arm B) were collected and performed with fluorescence-activated cell sorting (FACS) to sort eight pairs of VISTA + TAMs and VISTA − TAMs.After quality control (QC), three pairs of VISTA + TAMs and VISTA − TAMs were subsequently performed with smart-seq2.FDU-ZSH EXPC (Arm B) was our own dataset and contained 8 gastric cancer (GC) patients (Figure S1).(B and C) Differentially expressed genes (DEGs) Significance values were determined by paired t test (G [CD163, HLA-DR and CD11c] and H) and Wilcoxon matched-pairs signed rank test (G [CD206]).*p < .05,**p < .01,***p < .001and n.s.refers to not significant.F I G U R E 5 Blockade of V-domain immunoglobulin suppressor of T-cell activation (VISTA) reprograms tumour-associated macrophages (TAMs) to a proinflammatory phenotype.(A) Five fresh gastric cancer (GC) samples (Zhongshan Hospital Fudan University [FDU-ZSH] Experimental Cohort [EXPC] Arm D) were collected and digested into single-cell suspensions.The single-cell suspensions were incubated with IgG 2B (15 μg/mL, R&D Systems) or α-VISTA antibody (15 μg/mL, R&D Systems) in RPMI 1640 medium (Gibco) containing 10% foetal